1. Field of the Invention
The present invention generally relates to the field of molecular biology. More specifically, the invention relates to systems and methods for rapid identification and enrichment of nucleic acid aptamers that bind to specific target molecules. Disclosed herein is a method that may be employed to select aptamers based on susceptibility to digestion by nucleases. In part, this is accomplished by beginning with a nucleic acid library that forms predictable, complementary, secondary structure at the termini. The secondary structures contain sequences that can be digested by nucleases. The library is incubated with a target of interest, and the unbound molecules are digested with nucleases, whereas the bound molecules are afforded protection from digestion by nucleases. With each round of subtraction, the complexity of the initial library is reduced until only the desirable aptamers remain: those that bind a target of interest.
2. Description of the Related Art
Aptamers are nucleic acid molecules having specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing.
Aptamers, like peptides generated by phage display or monoclonal antibodies (MAbs), are capable of specifically binding to selected targets and, through binding, block their targets' ability to function. Created by an in vitro selection process from pools of random sequence oligonucleotides, aptamers have been generated for over 100 proteins including growth factors, transcription factors, enzymes, immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets (e.g., will typically not bind other proteins from the same gene family). A series of structural studies have shown that aptamers are capable of using the same types of binding interactions (hydrogen bonding, electrostatic complementarity, hydrophobic contacts, steric exclusion, etc.) that drive affinity and specificity in antibody-antigen complexes.
Aptamers have a number of desirable characteristics for use as therapeutics (and diagnostics) including high specificity and affinity, biological efficacy, and excellent pharmacokinetic properties. In addition, they offer specific competitive advantages over antibodies and other protein biologics, for example:
1) Speed and control. Aptamers are produced by an entirely in vitro process, allowing for the rapid generation of initial (therapeutic) leads. In vitro selection allows the specificity and affinity of the aptamer to be tightly controlled and allows the generation of leads against both toxic and non-immunogenic targets;
2) Toxicity and Immunogenicity. Aptamers as a class have demonstrated little or no toxicity or immunogenicity. In chronic dosing of rats or woodchucks with high levels of aptamer (10 mg/kg daily for 90 days), no toxicity is observed by any clinical, cellular, or biochemical measure. Whereas the efficacy of many monoclonal antibodies can be severely limited by immune response to antibodies themselves, it is extremely difficult to elicit antibodies to aptamers (most likely because aptamers cannot be presented by T-cells via the MHC and the immune response is generally trained not to recognize nucleic acid fragments;
3) Administration. Whereas all currently approved antibody therapeutics are administered by intravenous infusion (typically over 2-4 hours), aptamers can be administered by subcutaneous injection. This difference is primarily due to the comparatively low solubility and thus large volumes necessary for most therapeutic MAbs. With good solubility (>150 mg/ml) and comparatively low molecular weight (aptamer: 10-50 kDa; antibody: 150 kDa), a weekly dose of aptamer may be delivered by injection in a volume of less than 0.5 ml. Aptamer bioavailability via subcutaneous administration is >80% in monkey studies (Tucker et al., J. Chromatography B. 732: 203-212, 1999). In addition, the small size of aptamers allows them to penetrate into areas of conformational constrictions that do not allow for antibodies or antibody fragments to penetrate, presenting yet another advantage of aptamer-based therapeutics or prophylaxis;
4) Scalability and cost. Therapeutic aptamers are chemically synthesized and consequently can be readily scaled as needed to meet production demand. Whereas difficulties in scaling production are currently limiting the availability of some biologics and the capital cost of a large-scale protein production plant is enormous, a single large-scale synthesizer can produce upwards of 100 kg oligonucleotide per year and requires a relatively modest initial investment; and
5) Stability. Therapeutic aptamers are chemically robust. They are intrinsically adapted to regain activity following exposure to heat, denaturants, etc. and can be stored for extended periods (>1 yr) at room temperature as lyophilized powders. In contrast, antibodies must be stored refrigerated.
SELEX is a very powerful and novel technique used to search (i.e., pan) for aptamers exhibiting high-affinity and specific binding to target molecules of biomedical interest. SELEX searches a very large, random sequence oligonucleotide library (on the order of 1012 to 1015) for molecules exhibiting high-affinity binding to defined molecular targets. The small number of selected (i.e., high binding affinity) oligonucleotides can be geometrically amplified using the polymerase chain reaction (PCR) in a few weeks (or days with the use of robotics). Through a “survival of the fittest” sequential screening strategy (i.e., those having the highest binding affinity or catalytic activity) identification of novel aptamers can be accomplished rapidly in vitro using SELEX.
Despite its utility, SELEX is a laborious process requiring the physical separation of target bound aptamers and multiple rounds of sequential selection and amplification (Gold et al., 1995; Cox et al., 2002a, 2002b; Bruno and Kiel, 1999, 2002; Vivekananda and Kiel, 2006). The present invention seeks to exclude, by nuclease digestion, the number of candidate aptamers that are carried forward in sequential rounds of selection, thereby reducing the required number of selection rounds and increasing the likelihood that the surviving candidates are in fact aptamers that bind strongly to the target of interest.